加州鲈源型诺卡氏菌的分离鉴定及耐药性分析

Isolation, identification and drug resistance of Nocardia seriolae from Micropterus salmoides

为了确定引起河北省秦皇岛市3个养殖场加州鲈发病、大规模死亡的病原,试验共采集9尾加州鲈进行剖检,取结节内容物进行病原菌的分离、纯化及革兰氏染色、镜检,对分离菌进行生化鉴定、16S rDNA基因的序列分析、基因分型、毒力基因MCEIA检测、致病性试验和耐药性分析,并选择合适的抗生素对养殖场病鱼进行治疗,观察效果。结果表明:病死加州鲈肝脏、心脏、脾脏、肾脏和腹膜均有针尖大小的白色结节。从昌黎县、卢龙县和海港区3个养殖场的病死加州鲈体内各分离到1株优势菌,分别命名为CLY-1、LL-2和HG-3。3株分离菌在血琼脂培养基培养72 h均形成沙粒状、粗糙、易碎、边缘不整齐、有褶皱的淡黄色菌落,革兰氏染色、镜检均为紫红色分枝状的革兰氏阳性菌。3株分离菌均为过氧化酶阳性、氧化酶阴性,均能够发酵硝酸盐、七叶灵、柠檬酸,但不能利用酪素、黄嘌呤、酪氨酸、淀粉、明胶、山梨醇和甘露醇,符合诺卡氏菌的基本生化特性。3株分离菌16S rDNA基因测序结果完全一致,均与GenBank中的诺卡氏菌参考株16S rDNA基因相似性在99.9%以上,与鰤型诺卡氏菌参考株(ATCC 43993、NS128、JCM 3360、HSY-NS02、NParks_Aq54-21)亲缘关系较近,与杀鲑诺卡氏菌参考株(DSM 4490、DSM 44488、R89、DSM 40472、W30)和星形诺卡氏菌(LMB-7)亲缘关系较远,基因分型结果与鰤型诺卡氏菌相符,且均携带毒力基因MCEIA。接种CLY-1株的加州鲈于攻毒后第5天开始死亡,第14天死亡率高达80%,病鱼临床表现和剖检结果与3个养殖场患病加州鲈一致。3株分离菌对各抗菌药物的耐药性一致,均对多西环素、卡那霉素、新霉素、替米考星、氟苯尼考、复方新诺明和磺胺间甲氧嘧啶敏感,对青霉素、氨苄西林、头孢拉定和磺胺二甲氧嘧啶耐药。选择多西环素和氟苯尼考两种抗生素以拌料形式给药,迅速控制了疫情。说明3个养殖场加州鲈大规模死亡的原因均为鰤型诺卡氏菌感染,用多西环素和氟苯尼考治疗取得了明显效果。

In order to further identify the pathogen of Micropterus salmoidess'illness and death on a massive scale of three farms in Qinhuangdao City,Hebei Province,a total of 9 tail perca californica were collected from 3 farms for autopsy.The nodules were collected for isolation,purification,gram staining and microscopic examination of the pathogen.Biochemical identification,sequence analysis of 16S rDNA gene,genotyping identification,detection of virulence gene MCEIA,pathogenicity test and drug resistance analysis were also performed for the isolated bacteria,and select appropriate antibiotics to treat sick fish in farms and observe the effect.The results showed that white nodules of needle size were found in liver,heart,spleen,kidney and peritoneum.One dominant strain was isolated from each of the dead California perch from three farms in Changli County,Lulong County and Haigang district,and was named as Gly-1,LL-2 and HG-3,respectively.The 3 isolates were cultured in the blood AGAR medium for 72 h,and all of them formed sandy-grained rough and fragile light yellow colonies with irregular edges and folds.Gram staining and microscopic examination showed purplish red branching Gram-positive bacteria.The three isolates were both peroxidase positive and oxidase negative,and could fermentate nitrate,escherin and citric acid,but could not use casein,xanthine,tyrosine,starch,gelatin,sorbitol and mannitol,which was consistent with the basic biochemical characteristics of Nocardia.The results of 16S rDNA gene sequencing of the three isolates were completely consistent,and the genetic similarity between the three isolates and the Nocardia seriolae reference strain 16S rDNA in GenBank was more than 99.9%.Three isolates were related to the reference strain of Nocardia seriolae(ATCC 43993,NS128,JCM 3360,HSY-NS02,NParks_Aq54-21).Three isolates were far related to the reference strain of Nocardia salmonicides(DSM 4490,DSM 44488,R89,DSM 40472,W30)and Nocardia astroides(LMB-7),and the genotyping results were consistent with that of Nocardia seriolae,and both carried the virulence gene MCEIA.The mortality rate reached 80%on the 14th day after infection.The clinical manifestations and autopsy results of the infected fish were consistent with those of the three fish farms.The antimicrobial resistance of the three isolates was consistent,and they were sensitive to doxycycline,kanamycin,neomycin,Timicosin,flufenicol,cotrimoxazole and sulfamethoxil,and resistant to penicillin,ampicillin,cefradine and sulfamethoxil.Two antibiotics,doxycycline and flufenicol were selected to be administered in the form of mix,and the outbreak was quickly controlled.These results indicated that the cause of mass death of Micropterus salmoides in three farms was Nocardia seriolae infection,and the treatment with doxycycline and flufenicol was obvious.